Mucosal adjuvanticity and mucosal booster effect of colibactin-depleted probiotic Escherichia coli membrane vesicles.

There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (ΔflhDΔclbP). ΔflhDΔclbP-derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (ΔflhD-MVs). In addition, glycoengineered ΔflhDΔclbP-MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14+MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14+MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g. an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of ΔflhDΔclbP-MVs in the context of vaccine safety.

Iron Delivery through Membrane Vesicles in Corynebacterium glutamicum.

Bacterial cells form and release membrane vesicles (MVs) originating from cellular membranes. In recent years, many biological functions of bacterial MVs have been identified. Here, we show that MVs derived from Corynebacterium glutamicum, a model organism for mycolic acid-containing bacteria, can mediate iron acquisition and other phylogenetically related bacteria. Lipid/protein analysis and iron quantification assay indicate that C. glutamicum MVs formed by outer mycomembrane blebbing can load ferric iron (Fe3+) as its cargo. Iron-loaded C. glutamicum MVs promoted the growth of producer bacteria in iron-limited liquid media. MVs were received by C. glutamicum cells, suggesting a direct transfer of iron to the recipient cells. Cross-feeding of C. glutamicum MVs with phylogenetically close (Mycobacterium smegmatis and Rhodococcus erythropolis) or distant (Bacillus subtilis) bacteria indicated that C. glutamicum MVs could be received by the different species tested, while iron uptake is limited to M. smegmatis and R. erythropolis. In addition, our results indicate that iron loading on MVs in C. glutamicum does not depend on membrane-associated proteins or siderophores, which is different from what has been shown in other mycobacterial species. Our findings illustrate the biological importance of MV-associated extracellular iron for C. glutamicum growth and suggest its ecological impact on selected members of microbial communities. IMPORTANCE Iron is an essential element of life. Many bacteria have developed iron acquisition systems, such as siderophores, for external iron uptake. Corynebacterium glutamicum, a soil bacterium known for its potential for industrial applications, was shown to lack the ability to produce extracellular, low-molecular-weight iron carriers, and it remains elusive how this bacterium acquires iron. Here, we demonstrated that MVs released from C. glutamicum cells could act as extracellular iron carriers that mediate iron uptake. Although MV-associated proteins or siderophores have been shown to play critical roles in MV-mediated iron uptake by other mycobacterial species, the iron delivery through C. glutamicum MVs is not dependent on these factors. Moreover, our results suggest that there is an unidentified mechanism that determines the species specificity of MV-mediated iron acquisition. Our results further demonstrated the important role of MV-associated iron.

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

Nitric Oxide, an Old Molecule With Noble Functions in Pseudomonas aeruginosa Biology.

Pseudomonas aeruginosa, a Gram-negative bacterium, is characterized by its versatility that enables persistent survival under adverse conditions. It can grow on diverse energy sources and readily acquire resistance to antimicrobial agents. As an opportunistic human pathogen, it also causes chronic infections inside the anaerobic mucus airways of cystic fibrosis patients. As a strict respirer, P. aeruginosa can grow by anaerobic nitrate ( [Formula: see text] ) respiration. Nitric oxide (NO) produced as an intermediate during anaerobic respiration exerts many important effects on the biological characteristics of P. aeruginosa. This review provides information regarding (i) how P. aeruginosa grows by anaerobic respiration, (ii) mechanisms by which NO is produced under such growth, and (iii) bacterial adaptation to NO. We also review the clinical relevance of NO in the fitness of P. aeruginosa and the use of NO as a potential therapeutic for treating P. aeruginosa infection.

Environmental factors that shape biofilm formation.

Cells respond to the environment and alter gene expression. Recent studies have revealed the social aspects of bacterial life, such as biofilm formation. Biofilm formation is largely affected by the environment, and the mechanisms by which the gene expression of individual cells affects biofilm development have attracted interest. Environmental factors determine the cell's decision to form or leave a biofilm. In addition, the biofilm structure largely depends on the environment, implying that biofilms are shaped to adapt to local conditions. Second messengers such as cAMP and c-di-GMP are key factors that link environmental factors with gene regulation. Cell-to-cell communication is also an important factor in shaping the biofilm. In this short review, we will introduce the basics of biofilm formation and further discuss environmental factors that shape biofilm formation. Finally, the state-of-the-art tools that allow us investigate biofilms under various conditions are discussed.

Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum.

Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 µM) of nitrate (NO(-) 3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO(-) 3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 µM NO(-) 3 and 100 µM NO(-) 3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO(-) 3. Activation of denitrification could be achieved by supplementation with as little as 62.5 µM of NO(-) 3 according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts, and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO(-) 3 followed by a transient increase of NO(-) 2. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus.

A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

cAMP signaling affects irreversible attachment during biofilm formation by Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa responds to environmental changes and regulates its life cycle from planktonic to biofilm modes of growth. The control of cell attachment to surfaces is one of the critical processes that determine this transition. Environmental signals are typically relayed to the cytoplasm by second messenger systems. We here demonstrated that the second messenger, cAMP, regulated the attachment of cells. Our results suggest cAMP inhibited the transition from reversible to irreversible attachment. Further analyses revealed that cell surface hydrophobicity, one of the key factors in cell attachment, was altered by cAMP.